Evaluation of luteinizing hormone regulation of maturation and apoptosis, expression of LHR and FSHR in cumulus-oocyte complexes in Lanzhou fat-tailed sheep.

ABSTRACT

The present study aimed to assess LH effects on in vitro maturation (IVM) and apoptosis and also to explore the gene expressions of LHR and FSHR in cumulus-oocyte complexes (COCs) of the sheep. COCs were in vitro matured 24h in the IVM medium supplemented with varying concentrations of LH (0, 5, 10, 20 and 30 µg/mL). They were allocated into LH-1 (control group), LH-2, LH-3, LH-4 and LH-5 groups, respectively. FSH (10 IU/mL) addition was as a positive control (FSH group). COCs apoptosis was assessed by TUNEL. The qPCR and Western blotting were utilized to detect mRNA and protein expressions of FSHR and LHR, respectively. The results showed maturation rates of oocytes improved as LH concentration increased from 0 to 10 µg/mL (IU/mL), reaching a peak value of 44.3% in the LH-3 group. Maturation rate of LH-5 group was lower than that of LH-3 and FSH-treated groups. The lowest apoptosis rate was found in LH-3 group. The germinal vesicle break down (GVBD) rates of LH-2, LH-3 and LH-4 groups were also increased in comparison with that found in LH-1 group (control group). GVBD rate of LH-5 was lower than that in LH-3 group. The germinal vesicle (GV) rates in LH-3 and LH-4 groups were lower than those in LH-1 and LH-5 groups (p<0.05, or p<0.01). The lowest GV rate was found in LH-3 group. GV rates in LH-2, LH-4 and LH-5 groups were higher than that in FSH group (p<0.05). At hours 20, 22 and 24 after oocytes IVM, caspase-3 concentrations in four LH-treated groups were decreased in comparison with that in LH-1 group. At 24h, caspase-3 concentrations of LH-2 and LH-3 groups were lower than that in LH-1 group (p<0.05). Expression levels of FSHR and LHR mRNAs rose when LH concentrations in IVM medium increased. The greatest expressions of FSHR and LHR mRNAs were found in LH-5 and LH-3 groups (p<0.01) in comparison with those in the control group (LH-1). Meanwhile, FSHR mRNA expressions in LH-2, LH-3 and LH-4 groups were lower than that in FSH group (p<0.01 or p<0.05). Expression levels of FSHR proteins revealed no significant differences among all groups. Expression levels in LHR proteins were increased. LHR protein level in LH-2 group was higher than that in LH-1 group. In conclusion, LH treatment could promote the maturation rate and GVBD rate. LH reduced apoptosis rate, GV rate of sheep oocytes, and caspase-3 concentrations in IVM medium fluids and additionally enhanced expressions of FSHR and LHR mRNAs of sheep COCs.